Over the last fifty years, the incidence of obesity and its associated complications such as diabetes and heart disease has increased at alarming rates in developed nations (Kopelman, Nature, 2000, 404, 635-43; Seidell, Br. J. Nutr., 2000, 83, Suppl 1, S5-8; James et al., Obes. Res., 2001, 9, Suppl 4, 228S-233S; and Melanson et al., Cardiol. Rev., 2001, 9, 202-7). The level of obesity in a subject is a function of both adipose cell number and cell volume. The number of mature adipocytes is influenced by both the rate of differentiation of preadipocytes into adipocytes as well as adipocyte death by apoptosis. Obesity is often accompanied by glucose intolerance and insulin resistance in the liver, muscle and adipose tissue which can lead to defects in insulin secretion from pancreatic beta cells and eventually type II diabetes. Obesity is also associated with metabolic syndrome, hyperlypedemia and cardiovascular disease. Thus, the obesity epidemic has a profound effect on public health (Visscher et al., Annu. Rev. Public Health, 2001, 22, 355-75) and there is a great need for more therapuetics for the treatment or prevention of obesity (Van der Ploeg, Curr. Opin. Chem. Biol., 2000, 4, 452-60).
Adipogenesis is a complex process by which undifferentiated precursor cells differentiate, into fat cells. The peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, the largest family of transcription factors. Three distinct members of the PPAR subfamily have been described: alpha, delta (also called beta, NUC-1 or FAAR) and gamma. All of these PPAR gene family members are activated by naturally occurring fatty acids or fatty acid derivatives. PPARs heterodimerize with the retinoid X receptor (RXR) and regulate transcription of other genes through binding to specific PPAR recognition/response elements (PPREs), which consist of a direct repeat of the nuclear receptor hexameric DNA core recognition motif spaced by one nucleotide. A number of studies have reported that peroxisome proliferator activated receptor-gamma (PPAR-gamma; also known as PPAR-γ) plays a central role in glucose homeostasis and insulin sensitivity as well as adipogenesis. Furthermore, while cell proliferation and differentiation are sometimes considered to be mutually exclusive events, a close relationship has been established between these processes during adipocyte differentiation. One of the first events occurring during the adipogenesis program is re-entry of growth-arrested preadipocytes into the cell cycle following hormonal induction. After several rounds of clonal expansion, cells arrest proliferation again and undergo terminal adipocyte differentiation. PPAR-γ has been implicated in the control of cell proliferation and apoptosis as well as differentiation pathways in various malignancies, suggesting a role for PPAR-γ in carcinogenesis (Fajas et al., J. Mol. Endocrinol., 2001, 27, 1-9).
It has become clear that adipocytes play significant roles in regulating the body's metabolism (Bluher et al., Dev. Cell., 2002, 3, 25-38). The adipocyte, once thought of as a simple energy depot, is now known to be a highly specialized cell type involved in energy homeostatis, metabolic control and even behavior. In vivo, adipocyte differentiation is a complex process accompanied by coordinated changes in cell morphology, hormone sensitivity, gene expression and secretory capacity. Several transcription factors such as PPAR-γ, CCAAT/enhancer binding protein-alpha (CEBPα), and sterol-regulatory element-binding transcription factor 1 (SREBP1) are involved in this process (Spiegelman et al., Cell, 1996, 87, 377-89). Measurable changes occur during the progression of differentiation. These changes include the accumulation of triglycerides as lipid droplets, the secretion of several hormones and autocrine factors (e.g. leptin and adiponectin) and characteristic changes in gene expression including increased expression of PPAR-γ, hormone sensitive lipase (HSL), glucose transporter 4 (Glut4) and adipocyte lipid binding protein 2 (aP2). The process of adipocyte differentiation can be modeled in vitro by incubating pre-adipocytes with insulin, a PPAR-γ agonist, hydrocortisone and a compound that increases intracellular levels of cyclic adenosine monophosphate (cAMP), usually 3-isobutylmethylxanthine (IBMX).
Many experiments have shown that PPAR-γ appears to be an important regulator of adipocyte differentiation (Hamm et al., Ann. N.Y. Acad. Sci., 1999, 892, 134-45; Grimaldi, Prog. Lipid Res., 2001, 40, 269-81). In its role as a nuclear hormone receptor/transcription factor, PPAR-γ pairs with its heterodimeric partner, retinoid X receptor a (RXR-alpha), to form a DNA binding complex that regulates the transcription of adipocyte-specific genes (Kliewer et al., Proc. Natl. Acad. Sci. U.S.A., 1992, 89, 1448-52). PPARs possess the same P-box sequence (the DNA binding motif) of most type II steroid hormone receptors. To date, most PPREs are direct repeats with one intervening nucleotide (DR-1). RXR acts as the preferential binding partner and the PPAR/RXR heterodimer recognizes the PPRE (DiRenzo et al., Mol. Cell. Biol., 1997, 17, 2166-76). The elucidation of PPAR-γ-regulated genes and the pathways downstream of PPAR-γ is of fundamental importance in order to identify drug targets for the treatment of metabolic diseases. Currently, however, only limited information available about PPAR-γ downstream target genes exists.
In recent years, various data analysis techniques such as cluster analysis algorithms, have provided many possibilities to analyze microarray gene expression data that could group genes into co-regulatory clusters (Shannon et al., Pharmacogenomics, 2003, 4, 41-52). Similarly, promoter sequences of each gene in a cluster can be immediately fed to cis-regulatory discovery algorithms to identify motifs that share in functionally related genes (Keles et al., Bioinformatics, 2002, 18, 1167-75). Therefore, motifs that are common to a set of apparently co-expressed genes are plausible candidates for binding sites implicated in PPAR-γ transcriptional regulation. Several genes have already been characterized for the presence of PPAR-γ binding element. Though the PPREs possess the same P-box (TGACCTnTGACCT; SEQ ID NO:5) of type 11 steroid hormone receptors, the actual binding motif may differ compared to the consensus P-box element in PPAR-γ-regulated genes.
Currently, it is known that compounds, including thiazolidinediones, that bind and activate PPAR-γ can improve insulin sensitivity and reduce hyperglycemia and hyperlipidemia in man. Although it is still controversial, these drugs appear to increase insulin sensitivity in the adipose tissue, muscle and liver (Burant et al., J. Clin. Invest., 1997, 100, 2900-8). Thus, PPAR-γ agonists are becoming increasingly important in the fight against type II diabetes, and the market share of these drugs is increasing. However, PPAR-γ agonists have negative side effects; such negative side effects as unwanted weight gain and increased adipose mass confound the situation. Furthermore, the long-term effects of these drugs have yet to be determined.
There remains a long-felt need for compounds which modulate the expression of genes involved in adipogenesis, cell proliferation and/or cell differentiation, including candidate therapeutic agents and compounds useful in the treatment, attenuation or prevention of pathologies such as obesity, hyperlipidemia, atherosclerosis, atherogenesis, diabetes, hypertension, or other metabolic diseases as well as having potential applications in the maintenance of the pluripotent phenotype of stem or precursor cells. The present invention provides compounds and methods useful for modulating gene expression pathways, including methods relying on mechanisms of action such as RNA interference, small non-coding RNAs, dsRNA enzymes, antisense and non-antisense mechanisms. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify oligonucleotide compounds and methods for these uses.